Phenazine-1-carboxylic acid and soil moisture influence biofilm development and turnover of rhizobacterial biomass on wheat root surfaces.

Phenazine-1-carboxylic acid (PCA) is produced by rhizobacteria in dryland but not in irrigated wheat fields of the Pacific Northwest, USA. PCA promotes biofilm development in bacterial cultures and bacterial colonization of wheat rhizospheres. However, its impact upon biofilm development has not been demonstrated in the rhizosphere, where biofilms influence terrestrial carbon and nitrogen cycles with ramifications for crop and soil health. Furthermore, the relationships between soil moisture and the rates of PCA biosynthesis and degradation have not been established. In this study, expression of PCA biosynthesis genes was upregulated relative to background transcription, and persistence of PCA was slightly decreased in dryland relative to irrigated wheat rhizospheres. Biofilms in dryland rhizospheres inoculated with the PCA-producing (PCA+ ) strain Pseudomonas synxantha 2-79RN10 were more robust than those in rhizospheres inoculated with an isogenic PCA-deficient (PCA- ) mutant strain. This trend was reversed in irrigated rhizospheres. In dryland PCA+ rhizospheres, the turnover of 15 N-labelled rhizobacterial biomass was slower than in the PCA- and irrigated PCA+ treatments, and incorporation of bacterial 15 N into root cell walls was observed in multiple treatments. These results indicate that PCA promotes biofilm development in dryland rhizospheres, and likely influences crop nutrition and soil health in dryland wheat fields.

Long-Term Irrigation Affects the Dynamics and Activity of the Wheat Rhizosphere Microbiome.

The Inland Pacific Northwest (IPNW) encompasses 1. 6 million cropland hectares and is a major wheat-producing area in the western United States. The climate throughout the region is semi-arid, making the availability of water a significant challenge for IPNW agriculture. Much attention has been given to uncovering the effects of water stress on the physiology of wheat and the dynamics of its soilborne diseases. In contrast, the impact of soil moisture on the establishment and activity of microbial communities in the rhizosphere of dryland wheat remains poorly understood. We addressed this gap by conducting a three-year field study involving wheat grown in adjacent irrigated and dryland (rainfed) plots established in Lind, Washington State. We used deep amplicon sequencing of the V4 region of the 16S rRNA to characterize the responses of the wheat rhizosphere microbiome to overhead irrigation. We also characterized the population dynamics and activity of indigenous Phz+ rhizobacteria that produce the antibiotic phenazine-1-carboxylic acid (PCA) and contribute to the natural suppression of soilborne pathogens of wheat. Results of the study revealed that irrigation affected the Phz+ rhizobacteria adversely, which was evident from the significantly reduced plant colonization frequency, population size and levels of PCA in the field. The observed differences between irrigated and dryland plots were reproducible and amplified over the course of the study, thus identifying soil moisture as a critical abiotic factor that influences the dynamics, and activity of indigenous Phz+ communities. The three seasons of irrigation had a slight effect on the overall diversity within the rhizosphere microbiome but led to significant differences in the relative abundances of specific OTUs. In particular, irrigation differentially affected multiple groups of Bacteroidetes and Proteobacteria, including taxa with known plant growth-promoting activity. Analysis of environmental variables revealed that the separation between irrigated and dryland treatments was due to changes in the water potential (Ψm) and pH. In contrast, the temporal changes in the composition of the rhizosphere microbiome correlated with temperature and precipitation. In summary, our long-term study provides insights into how the availability of water in a semi-arid agroecosystem shapes the belowground wheat microbiome.

Accumulation of the antibiotic phenazine-1-carboxylic acid in the rhizosphere of dryland cereals.

Natural antibiotics are thought to function in the defense, fitness, competitiveness, biocontrol activity, communication, and gene regulation of microorganisms. However, the scale and quantitative aspects of antibiotic production in natural settings are poorly understood. We addressed these fundamental questions by assessing the geographic distribution of indigenous phenazine-producing (Phz(+)) Pseudomonas spp. and the accumulation of the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA) in the rhizosphere of wheat grown in the low-precipitation zone (<350 mm) of the Columbia Plateau and in adjacent, higher-precipitation areas. Plants were collected from 61 commercial wheat fields located within an area of about 22,000 km(2). Phz(+) Pseudomonas spp. were detected in all sampled fields, with mean population sizes ranging from log 3.2 to log 7.1 g(-1) (fresh weight) of roots. Linear regression analysis demonstrated a significant inverse relationship between annual precipitation and the proportion of plants colonized by Phz(+) Pseudomonas spp. (r(2) = 0.36, P = 0.0001). PCA was detected at up to nanomolar concentrations in the rhizosphere of plants from 26 of 29 fields that were selected for antibiotic quantitation. There was a direct relationship between the amount of PCA extracted from the rhizosphere and the population density of Phz(+) pseudomonads (r(2) = 0.46, P = 0.0006). This is the first demonstration of accumulation of significant quantities of a natural antibiotic across a terrestrial ecosystem. Our results strongly suggest that natural antibiotics can transiently accumulate in the plant rhizosphere in amounts sufficient not only for inter- and intraspecies signaling but also for the direct inhibition of sensitive organisms.

Biological control of take-all by fluorescent Pseudomonas spp. from Chinese wheat fields.

Take-all disease of wheat caused by the soilborne fungus Gaeumannomyces graminis var. tritici is one of the most important root diseases of wheat worldwide. Bacteria were isolated from winter wheat from irrigated and rainfed fields in Hebei and Jiangsu provinces in China, respectively. Samples from rhizosphere soil, roots, stems, and leaves were plated onto King's medium B agar and 553 isolates were selected. On the basis of in vitro tests, 105 isolates (19% of the total) inhibited G. graminis var. tritici and all were identified as Pseudomonas spp. by amplified ribosomal DNA restriction analysis. Based on biocontrol assays, 13 strains were selected for further analysis. All of them aggressively colonized the rhizosphere of wheat and suppressed take-all. Of the 13 strains, 3 (HC9-07, HC13-07, and JC14-07, all stem endophytes) had genes for the biosynthesis of phenazine-1-carboxylic acid (PCA) but none had genes for the production of 2,4-diacetylphloroglucinol, pyoluteorin, or pyrrolnitrin. High-pressure liquid chromatography (HPLC) analysis of 2-day-old cultures confirmed that HC9-07, HC13-07, and JC14-07 produced PCA but no other phenazines were detected. HPLC quantitative time-of-flight 2 mass-spectrometry analysis of extracts from roots of spring wheat colonized by HC9-07, HC13-07, or Pseudomonas fluorescens 2-79 demonstrated that all three strains produced PCA in the rhizosphere. Loss of PCA production by strain HC9-07 resulted in a loss of biocontrol activity. Analysis of DNA sequences within the key phenazine biosynthesis gene phzF and of 16S rDNA indicated that strains HC9-07, HC13-07, and JC14-07 were similar to the well-described PCA producer P. fluorescens 2-79. This is the first report of 2-79-like bacteria being isolated from Asia.

Transformation of Pseudomonas fluorescens with genes for biosynthesis of phenazine-1-carboxylic acid improves biocontrol of rhizoctonia root rot and in situ antibiotic production.

A seven-gene operon for the synthesis of phenazine-1-carboxylic acid was introduced into Pseudomonas fluorescens Q8r1-96, an aggressive root colonizer that produces 2,4-diacetylphloroglucinol and consistently suppresses take-all of wheat. The recombinant strains produced both antifungal metabolites and maintained population sizes comparable to those of Q8r1-96 over a seven-week period in the rhizosphere of wheat. The strains were no more suppressive of take-all or Pythium root rot than was Q8r1-96, but suppressed Rhizoctonia root rot at a dose of only 10(2) CFU per seed, one to two orders of magnitude lower than the dose of Q8r1-96 required for comparable disease control.